A one-step reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed and used for the detection of Cherry leaf roll virus (CLRV) and Strawberry latent ring spot virus (SLRSV). The protocol was used to test infected screen house plants and also plants from orchards and vineyards where the vector (Xiphinema diversicaudatum) of SLRSV was detected from the soil. The one-step RT-PCR protocol is rapid and sensitive and has the potential to be used for the diagnosis of CLRV and SLRSV in routine diagnostic laboratories.